Cellular change is often triggered by the binding of an extrinsic element, such as a ligand, to the extracellular domain of a cell surface membrane receptor protein. This binding can result in cellular changes by activating and controlling intracellular signaling cascades and cell regulatory mechanisms. As such, understanding the initial binding interaction between the ligand and its receptor protein can be of great interest to the scientific community. A greater understanding of this interaction would enable one skilled in the art to modulate the resulting signaling cascade governed by the ligand/receptor interaction by selecting agents for co-stimulation or inhibition of the binding of the ligand to its receptor.
The tumor necrosis factor (TNF) receptor family is a class of mammalian signaling molecules that play an important role in protection against infection and immune inflammatory responses such as cellular signal transduction, stimulation of cells to secrete cytokines, cytotoxic T cell proliferation, general cellular proliferation, lymph node formation, bone formation, and bone degradation. TNF-mediated cellular signaling often involves a molecular activation cascade, during which a receptor triggers a ligand-receptor mediated signal. Alterations in TNF activation can have profound effects on a multitude of cellular processes, such as the activation or inhibition of cell-specific responses, cell proliferation, inflammatory reactions, and cell death.
The interactions between TNF ligands and receptors may result in one-directional signaling (the interaction of the TNF receptor/ligand triggers a signaling cascade in the receptor only) or may result in bi-directional or reverse signaling. In the instances of bi-directional or reverse signaling, the interaction would not only activate the signaling cascade of the TNF receptor but would also trigger a signaling cascade in a cell bearing the TNF ligand. (S. Wiley et al., Jour. of Immun., 3235-39 (1996).) Thus, understanding the interaction between a TNF receptor and ligand may result in therapeutic treatments involving the inhibition or enhancement of either one or both of the TNF receptor activity or TNF ligand activity.
One member of the TNF receptor family is the transmembrane activator and CAML-interactor (TACI), a cell surface protein. The TACI protein has been isolated and is described in WO 98/39361. When activated, TACI stimulates the influx of calcium in lymphocytes and initiates the activation of a transcription factor through a combination of a Ca2+-dependent pathway and a Ca2+-independent pathway. Functions of TACI include controlling the response of lymphocytes to cancer and to foreign antigens in infections, graft rejection, and graft-vs.-host disease (GVHD). Furthermore, activation of lymphocyte signaling allows the positive selection of functional lymphocytes and negative selection against self-reactive clones. (WO 98/39361 at 15.)
TACI modulated signals are often activated by a extracellular ligand/receptor interaction, which then triggers an intracellular protein/protein interaction. One of the intracellular proteins which bind with the TACI protein has been identified. TACI interacts with the calcium-signal modulating cyclophilin ligand (CAML), a protein associated with the calcium pathway in lymphocytes. According to WO 98/39361, after the binding of the extracellular domain of TACI to an extracellular ligand, the cytoplasmic domain of TACI binds CAML, initiating a Ca2+-dependent activation pathway, which includes the activation of the transcription factors, NF-AT, AP-1 and NFkB, a factor implicated in the actions of other members of the TNF-receptor family. The regions for the interaction between TACI and CAML were defined as the cytoplasmic COOH-terminal 126 amino acids of TACI and the NH2-terminal 201 amino acids of CAML. CAML's ability to act as a signaling intermediate was verified by the inhibition of TACI-induced activation of the transcription factor when blocked by a dominant-negative mutant. (Von Büllow, G. et al., Science, Vol. 278, p.138-141 (1997).)
Although this interaction between the cytoplasmic domain of TACI and CAML has been identified, little is known about the extracellular ligand with which TACI interacts to initiate the intracellular cascades. Given the important role TACI plays in signal transduction and given the potential therapies that may arise from the manipulation of the signaling cascades, there is a need in the art for the identification and understanding of the interaction of TACI with its signaling ligand. Further, there is a need for the development of assays and therapeutic methods using the interaction between TACI and its signaling ligand.
Another TNF protein that has been recently discovered is a ligand that has been designated Neutrokine α, which is described in WO 98/18921. Identical nucleotide and polypeptide sequences have also been disclosed as “TL5” in EP 0869180A1 and as “63954” in WO 98/27114. As a member of the TNF family, Neutrokine α polypeptides were described as useful in the treatment of tumor and tumor metastasis, infections by bacteria, virus and other parasites, immunodeficiencies, inflammatory disease, lymphadenopathy, autoimmune diseases, and GVHD. Neutrokine α was also described as useful to mediate cell activation and proliferation. Further, Neutrokine α polypeptides were described as primary mediators of immune regulation and inflammatory response. (WO 98/18921 at 11; EP 0869180A1 at 3.)
As Neutrokine α polypeptides may inhibit immune cell functions, the ligand was described as also having a variety of anti-inflammatory activities. (WO 98/18921 at 49.) Specifically, it was said that Neutrokine α polypeptides could be used as an anti-neovascularizing agent to treat solid tumors and for other non-cancer indications in which blood vessel proliferation is not wanted. (Id.) The polypeptides could also be employed to enhance host defenses against resistant chronic and acute infections and to inhibit T-cell proliferation by the inhibition of IL-2 biosynthesis. Finally, Neutrokine α polypeptides could also be used to stimulate wound healing and to treat other fibrotic disorders. (Id.)
As such activities may be modulated by the Neutrokine α polypeptides, knowledge of how the ligand functions would be of significant interest to the scientific community. WO 98/18921, EP 0869180A1 and WO 98/27114, however, fail to identify specific receptors with which Neutrokine α polypeptides bind. Identification of the related TNF receptor would allow those skilled in the art to identify antagonists which may then be used in therapies to treat the disorders associated with the Neutrokine α polypeptides. Thus, there is a need to greater understand this TNF ligand, identify the receptors with which it interacts, and determine how the interaction functions.